Analysis of the immune response to a variety of infectious agents has been limited by the fact that it has often proved difficult to culture pathogens in quantities sufficient to permit the isolation of important cell surface antigens. The advent of molecular cloning has overcome some of these limitations by providing a means whereby gene products from pathogenic agents can be expressed in virtually unlimited quantities in a non-pathogenic form. Surface antigens from such viruses as influenza (1), foot and mouth disease (2), hepatitis (3), vesicular stomatitis virus (4), rabies (5), and herpes simplex viruses (6) have now been expressed in E. coli and S. cerevisiae, and, in the future, promise to provide improved subunit vaccines. It is clear, however, that the expression of surface antigens in lower organisms is not entirely satisfactory in that potentially significant antigenic determinants may be lost by virtue of incomplete processing (e.g., proteolysis, glycosylation) or by denaturation during the purification of the cloned gene product.
This is particularly true in the case of membrane proteins, which, because of hydrophobic transmembrane domains, tend to aggregate and become insoluble when expressed in E. coli. Cloned genes coding for membrane proteins can be expressed in mammalian cells where the host cell provides the factors necessary for proper processing, polypeptide folding, and incorporation into the cell membrane (7,8). While these studies show that membrane proteins can be expressed on the surface of a recombinant host cell, and, for example (8), that a truncated membrane protein lacking the hydrophobic carboxy-terminal domain can be slowly secreted from the host cell rather than be bound to it, it is not clear that either the membrane-bound protein thus expressed or the truncated protein thus secreted will be able to act, in fact, to raise antibodies effective against the pathogen from which the protein is derived.
Herpes Simplex Virus (HSV) is a large DNA virus which occurs in two related, but distinguishable, forms in human infections. At least four of the large number of virus-encoded proteins have been found to be glycosylated and present on the surface of both the virion and the infected cells (9). These glycoproteins, termed gA/B, gC, gD, and gE, are found in both HSV type 1 (HSV1) and HSV type 2 (HSV2), while in the case of HSV 2, an additional glycoprotein (gF) has been reported to be found (10). Although their functions remain somewhat of a mystery, these glycoproteins are undoubtedly involved in virus attachment to cells, cell fusion, and a variety of host immunological responses to virus infection (11). Although HSV 1 and HSV 2 show only ˜50 percent DNA sequence homology (12), the glycoproteins appear to be, for the most part, type-common. Thus, gA/B, gD, and gE show a large number of type-common antigenic determinants (13-16), while gC, which was previously thought to be completely type-specific (17,18), has also been found to possess some type-common determinants. Type-specific antigenic determinants can, however, be demonstrated using monoclonal antibodies for some of the glycoproteins (10,19), showing that some amino acid charges have occurred since HSV1 and HSV2 diverged.
One of the most important glycoproteins with respect to virus neutralization is gD (11). Considerable evidence has been adduced strongly suggesting that the respective gD proteins of HSV-1 and HSV-2 are related. For example, recombination mapping has localized the respective genes to colinear regions in both virus genomes. Amino acid analysis showed gross homology between the two proteins. The gD proteins induce neutralizing antibodies to both type 1 and type-2 viruses in a type-common manner (19-21). In addition, most monoclonal antibodies generated to these glycoproteins are type common, also suggesting a high degree of structural relatedness between the two types of glycoproteins (20). Some monoclonal antibodies, however, were found to react type-specifically, suggesting significant differences between the proteins (19). Peptide maps of the proteins also unambiguously revealed such differences (22a). These results although suggesting that these polypeptides are related, are insufficient to indicate exactly how close the relationship is.
In order to examine the nature of the type-commonality of HSV-1 and HSV-2 gD proteins, the DNA sequences of the gD genes from HSV1 and HSV2 were determined. The derived amino acid sequences showed similarity. The resultant derived protein sequences were also analyzed for structural differences by using a program designed to determine hydrophobic and hydrophilic regions of the protein. This analysis demonstrated a high degree of conservation on a gross structural level. Although several amino substitutions were found between the two glycoproteins, the vast majority of these substitutions were conservative, suggesting an important structural requirement of this glycoprotein to the virus.
In contrast to HSV-1, HSV-2 appears to encode yet another glycoprotein, termed gF (22b,10,22c,22d). Although the HSV-2 gF had an electrophoretic mobility which was much faster than HSV-1 gC, mapping studies with recombinant viruses revealed that this protein was encoded by a region of the HSV-2 genome which was approximately colinear with the gene for HSV-1 gC (22c, 22d). In addition, it has been recently demonstrated that a monoclonal antibody against HSV-2 gF will cross-react weakly with HSV-1 gC (22f) and that a polyclonal antiserum made against HSV-1 virion envelope proteins precipitated gF (22d), suggesting a possible structural homology between the two glycoproteins. Thus, it appeared that a possible homologue to HSV-1 gC was the HSV-2 gF protein. This relationship was investigated in accordance with the present invention.
To examine the relatedness between HSV-1 and HSV-2, it has been determined herein that a DNA sequence of a 2.29 kb region of the HSV-2 genome is colinear with the HSV-1 gC gene. Translation of a large open reading frame in this region demonstrates that a protein which has significant homology to HSV-1 gC is encoded in this region. It is suggested that this region encodes the HSV-2 gF gene and that the gF protein is the HSV-2 homologue of HSV-1 glycoprotein C.